The present invention relates to immunomodulating compositions, pharmaceutical compositions comprising the same, and the use of such compositions in the treatment of mammals. In particular, the compositions are directed to the treatment of diseases associated with immune system disorders.
Therapies are continuously being developed for the prophylaxis and treatment of cancer and autoimmune, infectious and inflammatory diseases, all of which may be a direct result of an inadequate immune system response. Some of these therapies attempt to use the immune system therapeutically.
One approach is based on the antigen-specific elements of the immune system, namely antibodies and T-cells. For example, research has been aimed at developing vaccines against foreign agents, or against certain endogenous chemical messengers, such as interleukins, to control or induce certain antibody reactions. A second approach is based on the isolation, cloning, expression and production of peptides and proteins from the nonantigen-specific parts of the immune system. For example, proteins, such as cytokines, which comprise the interleukins produced by white blood cells, and interferons, which stimulate lymphocytes and scavenger cells that digest foreign antigens, offer possibilities for therapies.
The treatment of cancer, for example, could be greatly enhanced if the early immune response to a tumor could be augmented so that the tumor does not reach a critical size. Strategies that have been suggested to augment the immune response to a tumor include: vaccines specific for tumor-associated antigens; the use of monoclonal antibodies against antigens on the surface of tumor cells, such as against the IL-2 receptor; the use of bispecific molecules containing antitumor antibodies and superantigens.
Relatively recently, the role of the physiologically active polypeptide, known as tumor necrosis factor (TNF), has been studied. In particular, TNF has been shown to induce necrosis of tumors, with no effect upon the normal tissues of the living body. The amino acid sequence of TNF, as well as the base sequence of the DNA coding for TNF, have been disclosed in U.S. Pat. No. 4,879,226.
Because TNF has been shown to have a role in inducing necrosis of tumors, any agent that can stimulate the production or bioavailability of TNF in vivo has potential utility as a treatment for various tumorous conditions. Additionally, any agent that can stimulate human monocytes and macrophages to produce TNF in vitro, is useful as a means for providing a source of TNF for therapeutic administration, as well as for analytical and diagnostic purposes.
Other diseases also have or involve an impaired immune system response. For example, autoimmune diseases are disorders in which the immune system produces an antibody against substances that are not foreign to the body, resulting in inflammation and consequent tissue damage. For example, rheumatoid arthritis (RA) is an autoimmune disease in which the body""s immune system mistakenly recognizes normal cells of the lining of joints, called synovium, as foreign. The autoimmune attack may destroy the lining completely. In the most severe cases, the joints cease to function and are replaced surgically with artificial joints. TNF is a mediator of the damage in RA. Progression from mild symptoms to severe disfigurement can be very rapid. As yet, no treatment is available for RA patients. Other essentially untreatable autoimmune diseases include lupus erythematosus, multiple sclerosis, and amyotrophic lateral sclerosis.
Infectious diseases, such as those caused by bacteria, virus, and other opportunistic pathogens, can only succeed by avoiding or defeating the body""s immune system. The immune system mounts or elicits either or both non-specific immune responses and specific immune response factors to fight such pathogens.
Non-specific immune responses are focused on cytokine production, principally by macrophages, and serve as a prelude to specific antibody responses. The inflammatory cytokines include TNF-xcex1 and mediate an acute response directed to the injury or infection sites, which is manifested by an increased blood supply. The pathogenic bacteria or viruses are engulfed by neutrophils and macrophages in an attempt to contain the infection to a small tissue space. Macrophages, therefore, play a key role in the defense against infectious diseases as follows:
(1) processing and presentation of antigens to lymphocytes so that antibody-mediated and cell-mediated immune responses can occur;
(2) secretion of cytokines central to immune response; and
(3) destruction of antibody-coated bacteria, tumor cells or host cells.
Macrophages can ingest and kill a wide variety of pathogens, such as bacteria, fungi, and protozoa (parasites). This ability is augmented when the macrophages are xe2x80x9cactivated.xe2x80x9d Secreted products of activated macrophages are more diverse than those from any other immune cell. These regulate both pro- and anti-inflammatory effects and regulate other cell types. These products include TNF-xcex1, IL-1xcex2, IL-6, hydrolytic enzymes, and products of oxidative metabolism Bacteria that are eliminated primarily through this cell-mediated immune process include tuberculosis and other related mycobacterial infections, such as atypical mycobacterial infections seen in up to 50% of AIDS patients, and anthrax, a potential bacteriological warfare agent. Fungal infections are common problems in immuno-suppressed patients, such as those afflicted with AIDS or organ transplant patients. Protozoa include organisms such as malaria.
Inflammatory diseases include endometriosis and inflammatory bowel disease, which also is mediated by immune processes. Endometriosis is an obscure disease of unknown cause and histogenesis that affects menstruating women. The disease is characterized by inappropriate implantation, growth, and function of endometrial cells. Endometrial cells and fragments, which are normally discharged during the menstrual cycle, are transported through the fallopian tubes into the peritoneal cavity where, in some women, they implant, proliferate, and develop into endometriotic lesions. However, because it appears the endometrial cells are present in the peritoneal cavity of all menstruating females, it is presently unclear why endometriosis develops in some, but not all, women. Endometriosis can result in painfully inflamed tissue, abnormal bleeding, widespread scaring, painful urination or defecation, and damage to a woman""s reproductive organs, even leading to infertility. No known treatment for endometriosis exists, short of pregnancy, which provides temporary relief, or surgery to remove the source of endometrial cells, which also causes sterility.
Recently, numerous reports have suggested that endometriosis is associated with changes in the immune system. Early reports indicate that immunosuppressive treatments are associated with an increase in endometriosis in rhesus monkeys. Since that time, alterations in both cell-mediated and humoral immunity have been observed in humans with endometriosis.
During the past several years, studies have focused on the role of macrophages in endometriosis. The underlying hypothesis for these studies was that the monocyte/macrophage system regulates endometrial cell growth and prevents proliferation of misplaced endometrial cells in normal, healthy women. In women with endometriosis, the misplaced endometrial cells are allowed to implant, giving origin to endometriosis. Development of endometriosis then is a stimulus to auto-antibody production against endometrial cells and cell-derived antigens. These auto-antibodies, together with products from activated macrophages, may then interfere with fertility and reproductive performance of affected women.
The cumulative results from these studies have revealed the following pertinent facts:
(1) the peritoneal disposal system (consisting primarily of macrophages) that is thought to be responsible for the destruction of ectopic endometrial cells within the peritoneal cavity may be defective in women with extensive endometriosis;
(2) the defective peritoneal macrophage activity in women with extensive endometriosis is related, at least in part, to a prostaglandin-mediated event. Thus, significant stimulation of peritoneal macrophages from women with extensive endometriosis in response to macrophage activators, such as gamma interferon and endotoxin, could only be achieved when a prostaglandin synthesis inhibitor was included in the activation culture;
(3) products of circulating monocytes of endometriosis patients may be directly involved in regulating the growth of endometrial cells. In an unique co-culture system, enhancement of autologous endometrial cell proliferation was seen with monocytes from the majority of endometriosis patients, while suppression of proliferation was seen with monocytes from the most fertile control patients; and
(4) the proliferation of endometrial cells from endometriosis patients can be modulated by macrophage-derived cytokines. The results obtained to date suggest that the proliferative response of endometrium from patients with limited disease can be enhanced by Interleukin-1-xcex2 (IL-1xcex2) and TNF-xcex1. In contrast, the proliferative response of endometrium from patients with extensive disease is suppressed by IL-1xcex2 and TNF-xcex1.
Accordingly, the results of these studies suggest that the function of monocytes and macrophages from endometriosis patients play a significant role in the pathophysiology of the disease. Moreover, it appears that some of these macrophage functions are differently affected by the severity of the disease. In women with limited disease, macrophages appear to be hyperactivated in the peritoneal cavity, and perhaps, in the circulation and their endometrial cells appear to be able to respond to different macrophage-derived growth factors. In contrast, extensive endometriosis is characterized by suppression of macrophage activation within the peritoneal cavity due, in part, to hypersecretion of immunoregulatory prostaglandins. Macrophage products also appear to regulate the proliferation of endometrium in women with extensive endometriosis; however, qualitative differences in the response of endometrium to different cytokines suggests that the consequences of defective macrophage activation in these women may contribute to or, perhaps, control the disease.
Inflammatory bowel disease (IBD) is a general term for a group of chronic inflammatory disorders of unknown etiology involving the gastrointestinal tract. These disorders include nonspecific ulcerative colitis and Crohn""s disease. Extraintestinal manifestations that may accompany these disorders (e.g., arthritis, pericholangitis) may represent autoimmune phenomena and therapeutic agents used to treat IBD, such as corticosteroids and azathioprine, may exert their effects via immunosuppressive mechanisms. Patients with inflammatory bowel disease may have humoral antibodies to colon cells, bacterial antigens such as Escherichia coli, lipopolysaccharide, and foreign proteins, such as cow milk protein. In general, the presence and titer of these antibodies does not correlate with disease activity; however, it is likely that these antigens gain access to immunocompetent cells secondary to epithelial damage. In addition, IBD has been described in association with agammaglobulinemia as well as IgA deficiency. Associated abnormalities of cell-mediated immunity include cutaneous anergy, diminished responsiveness to various mitogenic stimuli, and decreases in the number of peripheral T-cells.
Bile, which is secreted by the liver and stored in the gall bladder, has been investigated for various purposes, including the use of bile extracts to enhance bioavailability of drugs that are readily metabolized by normal liver function (see WO 90/12583) and to inhibit leucocytosis promotion in a mammal (see Shinoda et al., Chem. Pharm. Bull., 30, 4429-4434 (1982)). However, bile has never been considered to be a source of therapeutically useful compositions with respect to neoplastic, inflammatory or infectious diseases. Interestingly, in accordance with British Pat. No. 337,797, it was suggested to use the gall bladder, itself, as a potential source of anti-cancer agents, but only after the bile had been removed from the gall bladder, and the gall bladder thoroughly washed.
It has now been discovered that bile is an important source of a composition that can activate immune system cells, such as macrophages and monocytes, and is effective in treating various cancers, especially pancreatic cancer and malignant melanoma. In particular, it has been discovered that the composition of the present invention can stimulate TNF production both in vitro and in vivo from, for example, macrophages. This property may be useful in the treatment of infectious diseases.
It has also been discovered that the immunomodulating effect (especially the ability to down-regulate or suppress TNF-xcex1 production) of the present invention may also be useful in the treatment of other immune system-involved disorders, such as autoimmune diseases and inflammatory diseases and disorders.
The bile composition of the present invention is also believed to be useful as an adjuvant additive for vaccines directed to childhood diseases, and as a protection against rejection phenomena involved in xenograph procedures, for example.
The bile composition of the present invention is obtained by extraction of bile with a water-soluble or miscible solvent. The extract so obtained may be further processed to remove unnecessary or undesirable components therefrom.
The product obtained by the process of extracting bile disclosed in further detail hereinbelow has been found to have TNF-stimulating activity (or TNF-inhibitory activity-depending on the species source) and is believed to have activity against cancer, infections, autoimmune disorders, and inflammatory disorders. In particular, it is believed that the bile extract of the present invention is especially active against pancreatic and other cancers.
Obviously, the entire composition so obtained may not be necessary to obtain such activity. Accordingly, it is possible to further separate, fractionate, or otherwise process the product thus obtained, and still retain the desired ability to stimulate TNF production, for example, to act against the immune system disorders that underlie various diseases. Moreover, it is envisioned that it is possible to obtain synthetically a product with the same or similar ability to stimulate TNF production and act against immune system disorders. Thus, it is envisioned that the components of the product may be identified and analyzed as to their respective contributions to the desired characteristics of TNF stimulation and ability to act against immune system disorders, among other biological effects. Moreover, it is further envisioned that such identification and analysis will be used to manufacture a synthetic form of the product.
In one aspect, the present invention relates to a composition for use as an immunomodulator comprising small molecular weight components of less than 3000 daltons, and having one or more of the following properties:
a) is extractable from bile of animals;
b) is capable of stimulating monocytes and macrophages in vitro and in vivo;
c) is capable of modulating tumor necrosis factor production;
d) contains no measurable level of IL-1xcex1, IL-1xcex2, TNF, IL-6, IL-8, IL-4, GM-CSF or IFN-xcex3;
e) has an anti-proliferative effect in a malignant cell line;
f) shows no cytotoxicity to human peripheral blood mononuclear cells or lymphocytes; and
g) is not an endotoxin.
In accordance with a preferred embodiment, the composition is extracted from the bile of bovines and is capable of stimulating the release of TNF.
The composition of the invention may be prepared by (a) mixing bile from an animal, preferably a bovine, with a solvent that is soluble or miscible with water, preferably an alcohol, and preferably with an equal volume of an alcohol, to produce a bile/alcohol solution; (b) separating the solution which preferably is an alcohol-soluble fraction, and isolating therefrom a solution substantially free of alcohol, as by removing most of the alcohol, such as by the use of heat; (c) removing bile pigments from the solution to obtain a clear, yellowish liquid; (d) optionally treating the clear, yellowish liquid to substantially remove any residual alcohol; (e) removing fatty organic materials, as by extracting the clear, yellowish liquid with ether and isolating the aqueous phase; and (f) optionally removing residual ether from the aqueous phase.
The composition may be used without further modification by simply packaging it in vials and sterilizing. The composition may also be used in a concentrated form. A preferred concentrated form is prepared as follows. Prior to step (e) the clear, yellowish liquid may optionally be concentrated to about one-eighth of the volume of the bile/alcohol solution and after step (f) the aqueous phase may be concentrated so that it is one-tenth of the volume of the bile/ethanol solution.
The invention also relates to a pharmaceutical composition comprising the immunomodulating composition of the invention.
The invention further relates to a method of treating a patient comprising administering to said patient an effective amount of a composition of the invention. The invention still further relates to the use of a composition of the invention in the prophylaxis and treatment of diseases and conditions requiring modulation of the immune response; preferably infectious diseases, inflammatory diseases, autoimmune diseases, vaccination, rejection phenomena associated with xenographs, and neoplasias.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, reference is made herein to various publications, which are hereby incorporated by reference in their entirety.